Phage-dependent super-production of biologically active protein and peptides

ABSTRACT

This invention relates to a method for enhancing the production of biologically active proteins and peptides in bacterial cells by infecting bacterial cells of the producer strain, which contain a plasmid with one or more targeted genes, with bacteriophage λ with or without the targeted gene(s). The phage increases synthesis of the targeted protein and induces lysis of the producer strain cells. Super-production is achieved by cultivating the producer strain cells under culture conditions that delay lytic development of the phage. The biologically active proteins and peptides subsequently accumulate in a soluble form in the culture medium as the cells of the producer strain are lysed by the phage.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to recombinant DNA technology and moreparticularly to a new method for enhancing the production ofheterologous proteins in bacterial host cells. The disclosed methodinvolves infecting host cells, which contain plasmid encoding the geneof interest, with bacteriophage λ to induce lysis of the bacterial hostcells. Super-production may be achieved in selected host cells eitherwhen the plasmid alone carries at least one copy of the heterologous DNAor when both plasmid and phage λ each carry at least one copy of theheterologous DNA.

[0003] 2. Description of the Related Art

[0004] At present, genetic engineering methods allow creatingmicroorganism strains capable of producing substantial amounts ofvarious bioactive substances having important applications in medicineand industry. Typically, plasmid vectors into which a heterologous genehas been inserted are used to transform bacterial host cells. Differentstrains of E. coli are frequently used as recipient cells. Using suchplasmid-dependent transformation methods, E. coli cells have beenengineered to produce a variety of valuable human peptides and proteins,including insulin, γ-interferon, a number of interleukins,superoxidedismutase, plasminogen activator, tumor necrosis factor,erythropoietin, etc. These substances are either already used in medicalpractice or undergoing different stages of clinical studies.

[0005] However, the plasmid method has serious disadvantages. It istechnologically complicated, since the desired product has to beextracted from bacterial cells after biosynthesis, which is amulti-stage process. For example, interferon extraction involvesdisintegration of cells, buffer extraction, polyethylene-imineprocessing, clarification, precipitation by ammonium sulfate, dialysis,and centrifugation (Goeddel, EP 0043980). The necessity for suchextraction and purification steps not only complicates productiontechnology of the recombinant product, but also results in substantiallosses, especially during large-scale industrial production.

[0006] A further complicating factor is that at relatively high levelsof expression of the cloned genes, the eukaryotic proteins generatedtend to accumulate in the cytoplasm of E. coli as insoluble aggregates,which are often associated with cell membranes. Consequently, thealready difficult extraction and purification methods discussed abovemust be supplemented with additional technical procedures related to theextraction of the insoluble inclusion bodies. Usually, the insolubleproteins are solubilized using ionic detergents, such as SDS orlaurylsarcosine, at increased temperatures or in the presence ofdenaturants, such as 8 M urea or 6-8 M guanidine-HCl.

[0007] Often, the final stage of purification involves renaturation andreoxidation of the solubilized polypeptides, which is required torestore functional activity. Disulfide bonds, which are necessary forproper folding of the protein in its native conformation, must bereformed. Renaturation procedures, such as disulfide interchange, mayuse expensive and relatively toxic reagents, like glutathione, andoxidized 2-mercaptoethanol or dithiothreitol. Further, the final yieldof bioactive genetically-engineered proteins may be relatively low.Moreover, the presence of even trace concentrations of the toxicreagents needed to solubilize and then re-establish secondary andtertiary protein structure may prohibit subsequent clinical applicationof the proteins. Thus, the generation of targeted protein in the form ofinsoluble inclusion bodies within the bacterial host cells not onlycomplicates the production of recombinant proteins and results indiminished yield, but may also render the final protein unsuitable forclinical use (Fisher, B., Sumner, I., Goodenough, P. Biotech. andBioeng. 41:3-13, 1993).

[0008] The technological difficulties associated with the extraction ofproteins produced by the expression of heterologous genes fromplasmid-transformed bacterial host cells may be overcome by infectingthe transformed bacterial host cells with bacteriophage, whose lyticpathway results in lysis of the bearer cell. Thus, the desired proteinmay be simply released into the culture medium (Breeze A. S. GB 2 143238A). Accordingly, Breeze disclosed a method of increasing the yield ofenzyme produced in E. coli by infecting the bacterial cells with phage λcarrying a temperature-sensitive mutation in cI to provide controlledlysis. The cI-gene product is a repressor of early transcription andconsequently blocks transcription of the late region of the phage DNA,which is required for head and tail assembly and cell lysis (Mantiatis,T., Fritsch, E. F., Sambrook, J., MOLECULAR CLONING: A LABORATORYMANUAL, 1982, Cold Spring Harbor Laboratory Press). Bacteriophagescarrying a temperature-sensitive mutation in cI are able to establishand maintain the lysogenic state as long as the cells are propagated ata temperature that allows the cI-gene product to repress transcriptionof phage genes necessary for lytic growth. Accordingly, the transformedbacterial host cells may be cultivated at 30° C., wherein thecI-mediated suppression of phage DNA transcription continues and thephage remains in the lysogenic state, until the stage of maximum fermentproduction is reached. Subsequently, the culture temperature may beincreased to 42° C. for 30 minutes in order to inactivate the cIrepressor and permit the phage to begin its lytic development. The hostcells may then be incubated for 2-3 hours at 30° C. to allow completelysis and release of the enzyme (Breeze A. S. GB 2 143 238A).

[0009] Although Breeze teaches release of proteins from bacterialproducer cells, it requires cultivating producers at temperatures notexceeding 30° C., which is not the optimum temperature for growth of E.coli cells. Synthesis at the optimum temperature (37° C.) is notpossible, since cells at temperatures exceeding 32° C. undergo lysisbefore reaching the stage of maximum ferment accumulation due to thedevelopment of temperature-sensitive lytic prophage. Furthermore,incubation of the bacterial host cells at 42° C. for 30 min as disclosedby Breeze may activate proteases that destroy the targeted protein.

[0010] Auerbach et al. (U.S. Pat. No. 4,637,980) used a phage λ DNAfragment for inducing lytic release of recombinant products. In thatmethod, like Breeze, the temperature-sensitive mutation in λ cI-geneproduct was used to provide temperature-dependent lysis of the bacterialhost cells. The λ DNA fragment in Auerbach maintained functionalendolysin-encoding genes, N, Q, R and S, for producing lysozymefollowing inactivation of the cI repressor at 42° C. Most of theremaining phage genes were deleted; mutations in O and P genes preventedreplication of the phage DNA. Consequently, the λ DNA was not a fullyfunctional phage, capable of modulating expression of the targeted gene.Moreover, the λ DNA of Auerbach was not suitable for use as a vector forcarrying targeted genes. Further, as discussed above, incubation of thebacterial host cells at 42° to 44° C. for 90-120 min as disclosed byAuerbach may activate proteases that destroy the targeted protein.

[0011] In addition to providing for the lytic release of intact proteinfrom bacterial producer cells, bacteriophages have also been used as analternative to bacterial plasmid-based vectors, for carryingheterologous DNA into host bacterial cells. (Murray, N. E. and Murray,K., Nature 251:476-481, 1974; Moir, A., Brammar, W. J., Molec. gen.Genet. 149:87-99, 1976). Typically, amplification of genes and theirproducts is achieved during lytic growth of the phage, wherein the phagegenome is integrated into the bacterial host DNA (Panasenko, S. M.,Cameron, J. R., Davis, R. V., Lehman, L. R., Science 196:188-189, 1977;Murray, N. E. and Kelley, W. S., Molec. gen. Genet. 175:77-87, 1979;Walter, F., Siegel, M., Malke, H., 1990, DD 276,694; Mory, Y., Revel,M., Chen, L., Sheldon, I. F., Yuti-Chernajovsky, 1983, GB 2,103,222A).Usually, either lysogenic cultures of recombinant phage λ are used forinfecting the bacterial host cells, or “warmed up” bacterial cultures,already harboring recombinant lysogenic phage λ, are grown up to amplifyexpression of the heterologous genes.

[0012] Although there are examples of the successful use of λ vectorsfor expression of heterologous genes, λ vectors have been used primarilyfor gene cloning. Once cloned, the genes are transferred to plasmidvectors for more effective expression. For example, when E. coli isinfected by phage λ Charon 4A C15, containing the human β-interferongene, the quantity of interferon in cell lysate constituted 7-8×10⁶units/liter. After the DNA fragment bearing targeted gene was reclonedfrom phage to plasmid, β-interferon yield increased to 1×10⁸ units/liter(Moir, A., Brammar, W. J., Molec. gen. Genet. 149:87-99, 1976).

[0013] To increase the yield of heterologous protein generated inbacterial host cells by recombinant λ vectors, mutations in the phagegenome have been introduced that cause phage λ to lose its ability toinitiate bacterial cell lysis. Enhanced yield is thereby achieved byextending the period of time during which the heterologous gene isexpressed by the bacterial host cells. Thus, for example, the yield ofDNA ligase 1 in lysogenic cultures containing λ gt4ligS prophage, withamber-mutation in the S gene, was five times greater than the yield ofDNA ligase 1 in lysogenic cultures containing λ gt4lig prophage withoutthe amber-mutation (Panasenko, S. M., Cameron, J. R., Davis, R. V.,Lehman, L. R., Science 196:188-189, 1977). The phage λ S protein isrequired for lysis; therefore S⁻ mutants accumulate large numbers ofintracellular progeny phage particles, as well as the targeted protein,without lysing the host cells (Mantiatis, T., Fritsch, E. F., Sambrook,J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring HarborLaboratory Press).

[0014] Similar increases in the yield of DNA polymerase 1 were reportedfor lysogenic cultures containing recombinant phage λ withamber-mutations in the S and Q genes, compared to recombinant phage λwithout the amber-mutations (Murray, N. E. and Kelley, W. S., Molec.gen. Genet. 175:77-87, 1979). The phage λ Q protein is required fortranscription of the late region of the phage DNA, which includes manygenes involved in head and tail assembly and cell lysis. (Mantiatis, T.,Fritsch, E. F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL,1982, Cold Spring Harbor Laboratory Press).

[0015] In U.S. Pat. No. 4,710,463, Murray discloses lysogenizing anon-suppressing (Su°) strain of E. coli with phage λ containing thetemperature-sensitive mutation in cI, as well as mutations in λ S and Egenes. Consequently, prolonged cultivation of the lysogenic E. coli at37° C. leads to high levels of production of the recombinant protein,which is retained within the cells, since these are not lysed by phagegene products in the normal way, and since the recombinant phage genomeis not encapisdated, it remains available for transcription.

[0016] Despite the enhanced yield of heterologous proteins possibleusing λ-vectors with S and E mutations, the potential technicaladvantages of bacteriophage vectors related to the lytic release oftargeted proteins, may be lost with these mutations, because thetargeted proteins accumulate inside the bacterial cell. Thus, when alysis-defective mutant λ-vector is used for production of heterologousprotein, the extraction and purification steps, discussed above forbacterial cells transformed by plasmid vectors, along with the resultantlosses, must be performed.

SUMMARY OF THE INVENTION

[0017] The present invention discloses a method for producing abiologically active protein of interest. The method comprises the stepsof: (1) transforming a bacterial host cell with a plasmid having atleast one copy of an expressible gene encoding the protein, (2)infecting the transformed bacterial host cell with a bacteriophagecapable of mediating lysis and also capable of lytic growth withoutlysis, and (3) cultivating the bacterial host cell under a culturecondition that induces lytic growth of the cell without lysis until adesired level of production of the protein is reached.

[0018] In a preferred embodiment, the bacteriophage has atemperature-sensitive mutation. More preferably, the bacteriophage isbacteriophage λ and the temperature-sensitive mutation is cI₈₅₇. Theculture condition that induces lytic growth of the bacteriophage is at atemperature of greater than 32° C. Prior to the cultivating step, thebacterial host cells may be grown at a temperature, generally less thanabout 32° C. that prevents lytic growth of the bacteriophage.

[0019] In a variation of the disclosed method, the bacteriophage has amutation in at least one gene involved in bacteriophage-mediated lysisof the bacterial host cell. Preferably, the bacteriophage isbacteriophage λ and the at least one gene involved inbacteriophage-mediated lysis is selected from the group consisting of N,Q and R. Moreover, the bacterial host cell is preferably from a strainof E. coli. The strain of E. coli may or may not produce a suppressorfor the repair of amber-mutations.

[0020] Bacteriophage-mediated lysis of the bacterial host cell may bedelayed by culturing at higher multiplicities of infection compared tolower multiplicities of infection. The infecting bacteriophage may beprovided at a multiplicity of infection in a range of about 1 to about100 and more preferably, at a multiplicity of infection in a range ofabout 10 to about 25.

[0021] In another aspect of the present invention, the bacteriophage maycontain at least one copy of an expressible gene encoding the sameheterologous protein which is encoded by the plasmid.

[0022] A variation of the method for producing a biologically activeprotein in accordance with the present invention is disclosed. Themethod comprises the steps of: (1) transforming a bacterial host cellwith a plasmid having at least one copy of an expressible gene encodingthe protein, (2) infecting the transformed bacterial host cell with abacteriophage having at least one copy of an expressible gene encodingthe protein, and (3) cultivating the bacterial host cell under a culturecondition that allows expression of the plasmid and phage genes.

[0023] In accordance with another aspect of the present invention, abacterial host cell is disclosed. The bacterial host cell has a plasmidwith at least one copy of an expressible heterologous gene encoding aprotein, wherein the host cell is infected with a bacteriophage capableof mediating lysis and also capable of lytic growth without lysis.

[0024] The bacterial host cell preferably has a bacteriophage with atemperature-sensitive mutation. More preferably, the bacterial host cellis infected with bacteriophage λ and the temperature-sensitive mutationis cI₈₅₇.

[0025] In a variation of the bacterial host cell, the bacteriophage hasa mutation in at least one gene involved in bacteriophage-mediated lysisof the host cell. Preferably, the bacterial host cell is infected withbacteriophage λ having a mutation in at least one gene selected from thegroup consisting of N, Q and R. More preferably, the bacterial host cellis infected with bacteriophage λ with cI₈₅₇, Q_(am 117) and R_(am 54)mutations.

[0026] In a preferred embodiment of the bacterial host cell of thepresent invention, the host cell has a plasmid encoding a protein ofinterest and is also infected with a bacteriophage having at least onecopy of an expressible gene encoding the protein of interest.

[0027] The bacterial host cell in accordance with the present inventionis preferably a strain of E. coli. The strain of E. coli may or may nothave a suppressor for repairing amber-mutations. Similarly, the strainof E. coli may or may not be recA deficient. One preferred strain of E.coli host cells in accordance with the present invention contains aplasmid having at least one copy of an expressible heterologous geneencoding a protein, wherein the strain of E. coli is infected withbacteriophage λ having cI₈₅₇, Q_(am 117) and R_(am 54) mutations. Theprotein may be human alpha-2b interferon. More preferably, in additionto having a plasmid with at least one copy of a gene encoding a protein,the E. coli host cell also has a bacteriophage λ having cI₈₅₇,Q_(am 117) and R_(am 54) mutations and at least one copy of a geneencoding the protein. This bacteriophage preferably lacks a suppressorfor repairing amber-mutations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0028] Bacteriophage λ is useful as a vector because more than 40% ofthe viral genome is not essential for lytic growth. This area of the λgenome, located in the central region of the λ DNA, between genes J andN, may be replaced by heterologous DNA encoding a desired product. Thatregion is transcribed early during infection.

[0029] In order to maximize the expression of a targeted gene, whosesynthesis information is recorded in the area of phage's early genes,special conditions for the phage's development must be provided toensure proper replication. Further, transcription of the early area,containing the targeted gene, should be fostered, while transcription ofthe later genes, involved in cell lysis, should be decelerated. Thisslows down maturation of the λ particles and subsequent cell lysis.Consequently, the early phage products, including the targeted geneproduct, will accumulate in the bacterial cells. Deceleration of latetranscription, thereby extending expression of the targeted gene, may beaccomplished by: (1) mutations of phage genome that block expression ofthe later genes (2) increased multiplicity of infection, and/or (3)cultivation of the infected bacterial cells at a reduced temperature.

[0030] An important advantage of infecting producer cells with abacteriophage is that the phage causes a profound rearrangement of allmacromolecular synthesis in the bacterial host cells. By turning offtranscription of bacterial genes, phages may increase the copying of thetargeted gene, and consequently, increase the output of desired product.

[0031] In a preferred embodiment of the present super-production system,phage λ with amber-mutations that delay bacterial lysis (e.g., Q⁻ and R⁻mutations) are provided in a strain of E. coli, designated Su°, whichlacks the suppressor responsible for correcting amber-mutations in phageλ. In order to obtain a non-suppressing (Su°) strain of E. coli, Su°clones are selected from the wild-type Su⁺ population. Preferably, aselection marker is inserted into the phage DNA, e.g., tetracycline orampicillin resistance.

[0032] Selection of Bacterial Strains

[0033] Selection of non-suppressing (Su°) strains of E. coli, forexample, E. coli K 802 was carried out with phage λ cI₈₅₇ Nam7Nam53 blatet (hereinafter λ bla N′). Strain E. coli C600 (λ bla N′) served assource of the phage. This phage was obtained by insertion of plasmid pCV11 (bla tet) at EcoRI site into single-site (EcoRI) vector carryingts-mutation in repressor gene (cI₈₅₇) Then two amber-mutations wereintroduced into the phage N gene by recombination in vivo.

[0034] Clones were tested for non-lysogenicity with phage λ clear. Inaddition to phage λ bla N′, phage λ cI₈₅₇ Q_(am 117)R_(am 54) was usedto check for suppressor.

[0035] Media—Liquid nutrient media, LB and M9 as well as agar medium LBwere used for bacterial culture growth (Miller J. H., 1972, Experimentsin molecular genetics, Spring Cold Harbor, N.Y.).

[0036] Preparation of Phage Lysate—Lysogenic culture was grown in brothat 28° C. under intense aeration to a density of 2×10⁸ cells/ml followedby incubation at 43° C. for 20 min. Then it was kept at 37° C. underintense aeration. Cells were lysed in 60-80 min and phage was releasedinto the cultural medium. Phage titer was estimated by a conventionaltwo-layer technique. As a rule, 2×10¹⁰ PFU/ml of phage lysate wereobtained.

[0037] As is known, phage λ N′ mutant is not able to lyse the host cellsand is present in cells in the form of extremely unstable plasmids. Ifthe host cells contain suppressor, the amber-mutation is phenotypicallycorrected, the N protein is synthesized and the phage can developlytically. This difference in the viability of Su⁺ and Su° cells,infected by λ N′, is used as a basis for selection of spontaneouslyappearing Su° revertants from the E. coli Su⁺ cell population. Phage λwith an inserted plasmid that introduced the ampicillin and tetracyclineresistance markers into cells was used to prevent the nonlysing Su°cells from masking the search for mutants. The phage also containsts-mutation in the repressor gene that permits lytic development of suchphage resulting in cell lysis.

[0038] If the medium supplemented with ampicillin and tetracycline isinoculated with Su⁺ culture after its infection with phage λ bla N′ withsubsequent growth at 43° C., single suppressor-free cells containingphage λ bla N′ in the form of plasmids must develop on plates. Su°derivatives of the parent cultures are obtained by curing the cells fromthe phage. The method can be subdivided into several stages.

[0039] 1. Infection of Culture With Phage λ bla N′

[0040] The culture E. coli Su⁺ was grown on the M9 medium with maltoseat 37° C. under intense agitation to a density of 1-2×10⁸ cells/ml. Thecells were infected with phage λ bla N′ at a multiplicity of 5-10particles per cell and incubated for 20 min at 20° C. Under givenconditions, the infection efficiency is about 100%, in addition to thebulk of Su⁺ cells, the phage also infects single Su° cells.

[0041] 2. Selection of Suppressor-Free Cells Containing Marker Phage

[0042] After infection, cells were plated out on agar mediumsupplemented with 12 γ/ml tetracycline and 20 γ/ml ampicillin and grownat 43° C. In 24 h, single colonies developed, which were replated onagar medium with antibiotics and grown at 37° C.

[0043] 3. Curing of the Selected Clones From Phage λ bla N′

[0044] Since phage λ N′ in the E. coli Su° cells is in the form ofextremely unstable plasmids, in order to cure from the phage theselected clones were plated on selective agar medium without antibioticsand grown at 37° C. The number of cells that had lost the phage in thefirst passage on the medium without antibiotics amounted to 12-35%. Theselection of such cells was carried out by monitoring the loss ofantibiotic resistance and the acquisition of sensitivity to phage λclear.

[0045] 4. Testing of Cells for Repressor

[0046] The ability of phage λ with amber-mutations to form plaques onlawns of cured clones was checked. Isogenic suppressor-free derivativesof the parent E. coli Su⁺ strains are clones, on which phage λ bla N′did not form plaques, phage λ cI₈₅₇ Q_(am 117) R_(am 54) produced1-3×10⁵ PFU/ml, and phage λ cI₈₅₇ without mutations in genes Q and Rproduced 1×10¹⁰ PFU/ml.

[0047] Using this method, Su° revertants of E. coli K 802 Su⁺ wereobtained. Based on the cell number at the moment of infection and thenumber of Su° revertants among them, the frequency of occurrence ofsuppressor-free cells was about 3×10⁻⁷.

[0048] The use of suppressing (Su⁺) and non-suppressing (Su°) hoststrains together with phage λ to achieve super-production ofheterologous proteins and peptides may be more fully understood from thefollowing working examples.

EXAMPLE 1 Increased Synthesis of β-Lactamase in E. coli Transformed withpBR322 Carrying the β-Lactamase Gene (bla) following Infection by Phageλ

[0049] Bacterial cells, E. coli C-600 Su⁺, were transformed with plasmidpBR322-bla and cultivated in Aminopeptid medium (manufactured at theLeningrad meat processing and packing factory), diluted 1:1 in 0.15 MNaCl. The C-600 Su⁺/pBR322-bla transformants were then grown at 37° C.to a density of 1×10⁸ cells/ml and divided into three portions. Thecontrol portion was left intact. The second portion was infected withphage λ having the temperature-sensitive mutation in cI, designated λcI₈₅₇, and the third portion was infected with phage λ having thetemperature-sensitive mutation in cI, as well as amber-mutations in theQ and R genes, designated λ cI₈₅₇ Q_(am 117)R_(am 54). Phage mutationswere accomplished by standard recombinant method in vivo. Phagemultiplicity was approximately 10 phage bodies per 1 bacterial cell. Theλ-treated cultures were incubated for 15 min at 37° C. to inactivate thecI repressor, and then for 19 hr at 28° C. The control cultures wereincubated at 37° C. for the entire period. β-Lactamase activity wasdetermined by iodometric assay as described by Chaykovakaya, S. M. andVenkina, T. G. Antibiotics 7(5):453-456, 1962. A unit of activity isdefined as the minimum quantity of ferment necessary to inactivate1×10⁻⁷ M penicillin (60 units) in 1 hr at 37° C., pH 6.8-7.0. TABLE 1β-Lactamase Activity Bacterial Cell Culture (Units) 1 C-600Su⁺/pBR322-bla  833 2 C-600 Su⁺/pBR322-bla + λcI₈₅₇ 4400 3 C-600Su⁺/pBR322-bla + λcI₈₅₇Q_(am117)R_(am54) 8300

[0050] As shown in Table 1, β-lactamase synthesis in the C-600Su⁺/pBR322-bla cells infected by phage λ with mutations in the latergenes, Q and R, is almost 10 times greater than the synthesis in C-600Su⁺/pBR322-bla control cells.

EXAMPLE 2 Increased Output of β-Lactamase Encoded by bla-Gene Containedin Both Plasmid and Phage

[0051] Cultures of E coli W 3101 recA⁻13 Su° with and withouttransformation by pBR322-bla were cultivated in Aminopeptid mediumdiluted 1:1 with 0.15 M NaCl, at 37° C. to a density of 1×10⁸ cells/ml.A recA⁻ strain was used because these cells have a reduced ability toconduct recombination in areas of extended homology in both plasmid andphage (e.g. bla-gene). Thus, recA⁻ cultures avoid exclusion of thehomologous bla-gene. The cultures were divided into two portions. Thefirst portion, which was not exposed to phage, was incubated at 37° C.for 16 hr. The second portion was infected with phage λcI₅₈₇blaQ_(am 117)R_(am 54) at a multiplicity of about 10 phage bodiesper 1 bacterial cell and cultivated for 2.5-3 hr at 37° C., and then foran additional 14 hr at 28° C. β-Lactamase activity was measured by theiodometric method. The results, shown in Table 2 (below), are expressedin units, as defined above for Table 1.

[0052] Phage λ cI₅₈₇bla Q_(am 117)R_(am 54) was prepared from lysogeniccultures maintained at 28° C. in Aminopeptid medium. When the bacterialcell density reached about 1×10⁸ cells/ml, the cells were warmed for 20minutes at 43° C. in order to inactivate the cI repressor. Consequently,the prophage is excised from the bacterial genome and begins its lyticdevelopment. After 50 min, the cells underwent lysis, releasing 100-200bodies each. At a density of 1×10⁸ cells/ml, the cultures produced1-2×10¹⁰ phage bodies per ml. Thus, to infect bacterial cells with phageat a multiplicity of about 10 means that 1 ml of phage lysate (1×10¹⁰phage bodies) was added to 10 ml of bacterial suspension (1×10⁹ cells).TABLE 2 β-Lactamase Bacterial Cell Culture Activity (Units) 1 W 3101recA⁻13 Su°/pBR322-bla   13,555 2 W 3101 recA⁻13 Su° +λcI₈₅₇blaQ_(am117)R_(am54)   227,796 3 W 3101 recA⁻13 Su°/pBR322-bla +2,000,000 λcI₈₅₇blaQ_(am117)R_(am54)

[0053] As shown in Table 2, bacterial cells which were transformed withboth plasmid containing the targeted gene and phage carrying the samegene, produced about 10 times more recombinant protein (β-lactamase)than bacterial cells transformed with phage alone, and over 100 timesmore β-lactamase than bacterial cells transformed by plasmid alone.

EXAMPLE 3 Super-Production of the β-Galactosidase Encoded by lac-GeneContained in Both Plasmid and Phage

[0054] Cultures of E. coli RLM1 containing prophage λ cI₈₅₇ plac5Q_(am 117) R_(am 54) (carrying a copy of the β-galactosidase gene, lac5)were grown in LB medium (Difco) at 30° C. with intensive aeration to adensity of approximately 1×10⁸ cells/ml. The lysogenic culture waswarmed to 43° C. and incubated for 20 minutes to inactivate cIrepressor. The temperature was then decreased to 37° C. and thebacterial cells underwent lysis, with phages being formed at 1-2×10¹⁰PFU/ml. Subsequently, 10 liters of phage lysate, containing about 1×10¹⁰phage bodies (λ cI₈₅₇ plac5 Q_(am 117) R_(am 54)) per ml, were added to40 liters of a suspension of E Coli Ca 77 Su° transformed by plasmidpZ56 at a density of about 1×10⁸ cells/ml in LB medium. Thus, themultiplicity of infection was 25, i.e., there were 25 phage bodies perbacterial cell.

[0055] After 7 hr at 37° C., recombinant β-galactosidase constituted 1.9g per liter of culture medium. The activity of β-galactosidase wascalculated by the method of Miller (Miller, J. H., EXPERIMENTS INMOLECULAR GENETICS, 1972, Cold Spring Harbor Laboratory Press). A unitof activity was calculated as the minimum quantity of ferment requiredto hydrolyze 1 μM ortho-nitrophenyl-β-D-galactoside to orthonitrophenolper min at 30° C., pH 7.0.

EXAMPLE 4 Super-Expression of Human Interferon α-2b

[0056] Su⁺ and Su° strains of E. coli K 802, transformed with a plasmidbearing a single copy of the gene encoding alpha-2 human interferon(pIF-2-trp), were grown in LB medium to a density of 1.5-2×10⁸ cells/ml.These cells were then infected at a multiplicity of 15 with differentphage λ lysates, as indicated in Table 3 (below). Cultivation continuedwith intensive aeration at 25° C. for 13 hr. Control cultures, notinfected with phage, were incubated at 37° C. for the same period. TABLE3 Bacterial Interferon Cell Culture Phage (Units/L) 1 K 802-pIF-2-trpSu⁺  8.0 × 10⁷ 2 K 802-pIF-2-trp Su⁺ λcI₈₅₇  77 × 10⁷ 3 K 802-pIF-2-trpSu⁺ λcI₈₅₇Q_(am117)R_(am54)  340 × 10⁷ 4 K 802-pIF-2-trp Su⁺λ-pIF-2-trpcI₈₅₇Q_(am117)R_(am54) 1400 × 10⁷ 5 K 802-pIF-2-trp Su°λ-pIF-2-trpcI₈₅₇Q_(am117)R_(am54) 3000 × 10⁷

[0057] As shown in Table 3, when bacterial cells which were transformedwith plasmid containing the targeted gene were infected with phage λcontaining only the temperature-sensitive mutation in cI, interferonexpression increased by about 10-fold compared to control, non-infectedcultures. Adding the amber-mutations in Q and R genes further increasedexpression by about 40-fold compared to control bacterial cells. Addinga copy of the interferon gene to phage λ with cI, Q and R mutationsincreased interferon synthesis by 175-fold over controls. Finally, whena non-suppressing, Su°, strain of E. coli, transformed by a plasmidbearing a copy of the interferon gene, was infected with phage λ, alsohaving a copy of the interferon gene, as well as cI, Q and R mutations,the bacterial host cells produced about 375 times more recombinantprotein than the control cells transformed by plasmid alone.

EXAMPLE 5 Enhanced Recovery of Biologically Active RecombinantInterferon by Phage-Mediated Host Cell Lysis

[0058] Strain E. Coli SG 20050 was transformed by a plasmid bearing twocopies of the human interferon alpha-2b gene (plF-14) by standardmethods. The transformant cells were grown up in 80 liters of LB mediumat 37° C. with intensive aeration to a density of 2×10⁸ cells/ml. Theculture was divided into two portions. The first was not infected withphage. The second was infected with phage λ lysate harvested from E.coli K 802/λ cI₈₅₇ Q_(am 117) R_(am 54) at a multiplicity of 10 phagebodies per bacterial cell. The control cells were incubated for 19 hr at37° C. and the phage-infected cells were incubated for 19 hr at 21° C.

[0059] Interferon production in both control and phage-infected cultureswas about 20% of the total cellular protein. However, the interferon incontrol cells was associated at least in part with insoluble inclusionbodies. Thus, it was not possible to determine its biological activitywithout solubilization, denaturation and renaturation. In contrast, thespecific activity of the soluble interferon released into the mediumfollowing phage-mediated cell lysis, was readily determined by standardimmunoenzyme assay. The interferon activity was 4×10¹⁰ IU/liter (200mg/liter).

[0060] Pre-clinical toxicological studies of recombinant human alpha-2βinterferon produced by the phage super-production method of the presentinvention showed that the compound was practically non-toxic.Intra-abdominal and intramuscular injections of the recombinantinterferon in white mice and Wistar rats at 8.5×10⁹ ME/kg (2.5×10⁵ timesthe maximum human dose) and intravenous injections in mice and rabbitsat 4.25×10⁹ ME/kg (1.25×10⁵ times the human therapeutic dose) producedno pronounced intoxication or death of the animals. Four months ofinjections in Wistar rats at 6×10⁵, 6×10⁶ and 3×10⁷ ME/kg (18, 180 and900 times the human therapeutic dose, respectively) showed no damage tothe main organs and bodily systems of the experimental animals.Likewise, three months of intravenous injections in rabbits of 6×10⁵ and6×10⁶ ME/kg, and two months of intramuscular injections in dogs at 3×10⁶ME/kg showed no signs of damage to the organ systems.

[0061] During immunotoxic and allergenic analysis of recombinantinterferon, the induction of cellular and humoral immunity reactions, aswell as delayed and immediate hypersensitivity reactions were studied.The results indicated that no immunotoxic or allergenic influence wasproduced. The recombinant interferon also had no mutagenic orDNA-damaging effects in bacteria during metabolic activation in vitro orin bone marrow of mouse embryos in vivo.

[0062] Embryotoxic studies of recombinant interferon were conducted inpregnant hamadryad baboon females. Daily intramuscular doses duringorganogenesis (20^(th) to 50^(th) days of pregnancy) caused defects inembryo development leading to miscarriage or stillbirth. Similar resultswere obtained for recombinant interferon analogs, and most probablycould be explained by a powerful antiproliferative action ofinterferons. It is possible that the miscarriage may be attributed to a“cancellation” of immunologic tolerance of maternal organism towards thefetus, caused by immuno-modulating action of the protein.

[0063] Recombinant interferon was also studied in Ukrainian clinics.Based on these clinical studies, the recombinant interferon was shown tobe useful in the treatment of a variety of human diseases andconditions. For example, recombinant interferon was effective intreating acute and chronic hepatitis B, acute viral, bacterial and mixedinfections, acute and chronic septic diseases, herpetic infections,herpes zoster, papillomatosis of larynx, multiple sclerosis, and variouscancers, including melanoma, renal cell carcinoma, bladder carcinoma,ovarian carcinoma, breast cancer, Kaposi's sarcoma and myeloma.

[0064] The contraindications in human clinical applications wereprolonged (several months) use at high doses, allergy and pregnancy. Thepossible side effects noted were small and transitory “flu-like”symptoms and at prolonged regimens, leuko and trombocytopenia weremarked.

[0065] While we have described a number of embodiments of thisinvention, it is apparent that our description of the invention can bealtered to provide other embodiments that utilize the basic process ofthis invention. Therefore, it will be appreciated by those of skill inthe art that the scope of this invention is to be defined by the claimsappended hereto rather than the specific embodiments that have beendescribed in detail above by way of example.

What is claimed is:
 1. A method for producing a biologically activeprotein, comprising: transforming a bacterial host cell with a plasmidhaving at least one copy of an expressible gene encoding said protein;infecting the transformed bacterial host cell with a bacteriophagecapable of mediating lysis and also capable of lytic growth withoutlysis; and cultivating the bacterial host cell under a culture conditionthat induces lytic growth of said cell without lysis until a desiredlevel of production of said protein is reached.
 2. The method of claim 1, wherein the bacteriophage has a temperature-sensitive mutation.
 3. Themethod of claim 2 , wherein the bacteriophage is bacteriophage λ and thetemperature-sensitive mutation is cI₈₅₇.
 4. The method of claim 2 ,wherein said culture condition that induces lytic growth of thebacteriophage is at a temperature of greater than 32° C.
 5. The methodof claim 2 , wherein prior to the cultivating step, the bacterial hostcells are grown at a temperature which prevents lytic growth of thebacteriophage.
 6. The method of claim 5 , wherein the temperature whichprevents lytic growth of the bacteriophage is less than about 32° C. 7.The method of claim 1 , wherein the bacteriophage has a mutation in atleast one gene involved in bacteriophage-mediated lysis of the bacterialhost cell.
 8. The method of claim 7 , wherein the bacteriophage isbacteriophage λ and the at least one gene involved inbacteriophage-mediated lysis is selected from the group consisting of N,Q and R.
 9. The method of claim 1 , wherein the bacterial host cell is astrain of E. coli.
 10. The method of claim 9 , wherein the strain of E.coli produces a suppressor for the repair of amber-mutations.
 11. Themethod of claim 9 , wherein the strain of E. coli lacks a suppressor forthe repair of amber-mutations.
 12. The method of claim 1 , wherein theinfecting bacteriophage is provided at a multiplicity of infection in arange of about 1 to about
 100. 13. The method of claim 1 , wherein theinfecting bacteriophage is provided at a multiplicity of infection in arange of about 10 to about
 25. 14. The method of claim 1 , whereinbacteriophage-mediated lysis of the bacterial host cell is delayed athigher multiplicities of infection relative to lower multiplicities ofinfection.
 15. The method of claim 1 , wherein the bacteriophagecontains at least one copy of an expressible gene encoding said protein.16. A method for producing a biologically active protein, comprising:transforming a bacterial host cell with a plasmid having at least onecopy of an expressible gene encoding said protein; infecting thetransformed bacterial host cell with a bacteriophage having at least onecopy of an expressible gene encoding said protein; and cultivating thebacterial host cell under a culture condition that allows expression ofsaid genes.
 17. The method of claim 16 , wherein the bacteriophage has atemperature-sensitive mutation.
 18. The method of claim 17 , wherein thebacteriophage is bacteriophage λ and the temperature-sensitive mutationis cI₈₅₇.
 19. The method of claim 16 , wherein the bacteriophage has amutation in at least one gene involved in bacteriophage-mediated lysisof the bacterial host cell.
 20. The method of claim 19 , wherein thebacteriophage is bacteriophage λ and the at least one gene involved inbacteriophage-mediated lysis is selected from the group consisting of N,Q and R.
 21. The method of claim 16 , wherein the bacterial host cell isa strain of E. coli.
 22. The method of claim 21 , wherein the strain ofE. coli produces a suppressor for repairing amber-mutations.
 23. Themethod of claim 21 , wherein the strain of E. coli lacks a suppressorfor repairing amber-mutations.
 24. A bacterial host cell with a plasmidhaving at least one copy of an expressible heterologous gene encoding aprotein, wherein said host cell is infected with a bacteriophage capableof mediating lysis and also capable of lytic growth without lysis. 25.The bacterial host cell of claim 24 , wherein the bacteriophage has atemperature-sensitive mutation.
 26. The bacterial host cell of claim 25, wherein the bacteriophage is bacteriophage λ and thetemperature-sensitive mutation is cI₈₅₇.
 27. The bacterial host cell ofclaim 24 , wherein the bacteriophage has a mutation in at least one geneinvolved in bacteriophage-mediated lysis of the host cell.
 28. Thebacterial host cell of claim 27 , wherein the bacteriophage isbacteriophage λ and the at least one gene involved in bacteriophagemediated lysis is selected from the group consisting of N, Q and R. 29.The bacterial host cell of claim 24 , wherein the bacteriophage isbacteriophage λ having cI₈₅₇, Q_(am 117) and R_(am 54) mutations. 30.The bacterial host cell of claim 24 , wherein the bacteriophage has atleast one copy of an expressible heterologous gene encoding saidprotein.
 31. The bacterial host cell of claim 24 , wherein the bacterialhost cell is a strain of E. coli.
 32. The bacterial host cell of claim31 , wherein the strain of E. coli lacks a suppressor for repairingamber-mutations.
 33. The bacterial host cell of claim 31 , wherein thestrain of E. coli is recA deficient.
 34. A strain of E. coli with aplasmid having at least one copy of an expressible heterologous geneencoding a protein, wherein said strain of E. coli is infected withbacteriophage λ having cI₈₅₇, Q_(am 117) and R_(am 54) mutations. 35.The strain of claim 34 , wherein said protein is human alpha-2binterferon.
 36. The strain of claim 34 , wherein said strain of E. colilacks a suppressor for repairing amber-mutations.
 37. The strain ofclaim 36 , further comprising recA-13.
 38. A strain of E. coli with aplasmid having at least one copy of an expressible heterologous geneencoding a protein, wherein said strain of E. coli is infected withbacteriophage λ having cI₈₅₇, Q_(am 117) and R_(am 54) mutations and atleast one copy of an expressible heterologous gene encoding saidprotein.
 39. The strain of claim 38 , wherein said strain of E. colilacks a suppressor for repairing amber-mutations.
 40. The strain ofclaim 37 , wherein said protein is human alpha-2b interferon.